Stem etiolation has been successfully used as a pretreatment in cutting propagation. The technique involves forcing new shoot growth under conditions of heavy shade or total darkness and then using this growth as the cutting propagule.
Banding is a pretreatment, adjunct to etiolation, which involves excluding light from that zone of the stem which is to become the cutting base. Banding may either be applied to etiolated shoots which are subsequently allowed to turn green in the light, or applied to lightgrown developing shoots which are still in the softwood stage, in which case the band is said to "blanch" the underlying tissues. The response to etiolation and banding are, in effect, the same in that the tissues shielded from light are characteristically chlorotic, and maintained in a soft or succulent condition.
The earliest reports of etiolation as a pretreatment to propagation by cuttings came in the 1920's. Black paper coverings were used to produce etiolated cuttings of camphor (Reid, Trans. and Proc. Bot. Soc. Edinb., 28:184-188 (1922); Blackie et al, Kew Bulletin, pp 380-381 (1926)) and clematis (Smith, Trans. and Proc. Bot. Soc. Edinb., 29:17-26 (1924)), which had not previously been known to root adventitiously. In 1936 Gardner referred to etiolation as a method of some practicality. His work with apple and other species was the first mention of etiolation and banding being used together to facilitate the rooting of difficult-to-root species. By wrapping black insulation tape close to the growing point, thus blanching the cuttings while they were still attached to the tree, he increased rooting by 70%. He also built light tight boxes in which to pre-etiolate shoots. These shoots were then banded for up to a year. After that time the cuttings of apple rooted at 98%. The response to etiolation plus banding varied among apple varieties, although in all instances it increased rooting. Gardner also made the first mention of root primordia, up to 1/2" in length, being produced under banded stems still on the tree. (See Gardner, Pro. Amer. Soc. Hort. Sci., 34:323-329 (1936)).
Knight et al, J. Pomol., 6:47-60 (1937) compared etiolation under black cloth with stooling as pretreatments to the propagation of plum tree stock. They found that etiolated cuttings rooted better, but were also more susceptable to infections of Botrytis, while stooled cuttings also rooted well, but suffered no losses to Botrytis.
The first physiological studies of the effects of light on rooting and plant morphogenesis came from A. W. Galston and coworkers (Galston et al, Amer. J. Bot.. 36:85-94 (1949); Galston et al, Amer. J. Bot., 40:512-516 (1953): Galston et al, (1961), "Light and Life", W. D. McElroy and B. Glass (ed.). John Hopkins Press, Baltimore. pp 687-705). Galston et al, (1949 supra) studied the interaction of auxin with light and plant growth, noting that auxins profoundly influenced the growth and form of plants in minute amounts. They postulated that light affects morphogenesis by the alteration of endogenous auxin levels. Possible events affected by light included auxin synthesis, transport, photodestruction in intense light, the equilibrium of bound and free forms of auxin, and even changes in the activity of indole acetic acid (IAA) inactivating enzymes. They thought that light might also alter the responses of cells to auxin without affecting auxin levels. This might be caused by changing cell properties and affecting growth systems unrelated to auxin, thereby decreasing the apparent effectiveness of auxins. Light might even affect the synthesis of anti-auxins or nonspecific growth inhibitors (Galston et al, (1949 supra)). Attempts were made to separate these possible effects of light using a system which measured the growth of Pisum sativum `Alaska`. Seedlings in this system were grown in the dark and then either maintained in darkness or exposed to light. They concluded that the effect of light was to decrease the tissue responsiveness to both endogenous and applied auxin (Galston et al, (1949 supra)).
Galston et al, (1953 supra) made note of the fact that the action spectrum for light effects on growth responses to auxin was nearly identical to that reported for photoperiodsensitive plants and set out to investigate the nature and kinetics of red-light induced growth responses to auxin in etiolated peas. They conjectured that light altered the activity of an enzyme involved in the regulation of auxin metabolism. Possibilities included the indole acetic acid oxidase (IAA-0) system, and catalase, which prevents the destruction of IAA by IAA-0. Both these systems were known to be markedly affected by visible radiation (Galston et al, (1953 supra)).
A later report by Galston et al, (1961 supra) listed some of the striking differences in morphology and physiology between light grown and dark grown pea stems (Table 1), suggesting that normal auxin relationships differ in the two forms.
Shapiro, (1958), K. V. Thimann (ed.), "The Physiology of Forest Trees". Ronald Press, N.Y. pp 443-465, used lombardy poplar as a model system to investigate the effect of light on the growth of dormant root primordia, found in the phloem of two year old stems as unorganized groups of meristematic cells. Shapiro determined that these root primordia were sensitive to red over far-red light, implicating the involvement of a phytochrome type photoreceptor. Shapiro also noted that banding stems induced root primordia growth in the shaded areas, but that this would not promote root growth in adjacent lighted areas. Thus Shapiro hypothesized that there were normobile factors affecting root growth which were influenced by light.
TABLE 1 ______________________________________ Morphological differences between etiolated and light grown tissues. FACTOR ETIOLATED LIGHT GROWN ______________________________________ color white green internode length v. long shorter nutrition heterotrophic auto or heterotrophic sucrose opt. .about.2% .about.1% IAA opt. .about.10.sup.-6 M .about.10.sup.-4 M IAA-O activ. high low IAA-O inhib. absent or low high ______________________________________
The years from 1960 to the present saw numerous important contributions to the understanding of both the use and mechanism of light exclusion in the promotion of root initiation and rootgrowth. Noteworthy reviews include that by Frolich, Proc. Int. Plant Prop. Soc., 11:277-283 (1961), on work preceding this period, Hess, (1969), pp. 42-53, in "Root Growth", (ed.), W. J. Whittington. Butterworths and Co. LTD., London, on the factors affecting root initiation, and Ryan, Proc. Int. Plant Prop. Soc., 19:69-71 (1969), on etiolation effects on rooting. Hess confirmed that light plays a primary role among external influences on rooting, acting, Hess suggested, to improve rooting through a decrease in the lignification of the stem, and accompanying increases in free phenolic acid compounds. The latter acting directly as auxin cofactors to induce rooting, or as
degrading enzymes (Hess, (1969 supra)). Ryan, (1969 supra) reported that the lack of chlorophyll characteristic of etiolated tissues was not likely to be of consequence to rooting, as etiolated shoots were known to green quickly in the light while the benefit of the etiolation declined gradually. Ryan also made the wise observation that in some plants rooting shows no sensitivity to light, as in philodendron, or juvenile forms of Hedera helix. The bulk of the work cited by Ryan verified that light inhibits rooting and root elongation, whether by light mediated changes in stem anatomy, cofactor levels, carbohydrates, or auxin status. In his summation of the previous 40 years of research on light and rooting Ryan stated that at that point, "with our present knowledge of the rooting response to exclusion of light, the propagator can continue to make use of the etiolation effect on otherwise difficult to propagate plants without knowing why it is so effective." (Ryan, (1969 supra)).
Practical investigations which have been undertaken to evaluate the etiolation, shading, and blanching of shoots as a pretreatment to cutting propagation are described in the above references as well as in the following: The beneficial effects of etiolation and banding have been reviewed a number of times (Frolich, (1961 supra); Herman et al, Proc. Int. Plant Prop. Soc., 13:42-62 (1963); Kawase, Physiol. Plant., 18:1066-176 (1965); Ryan, (1969 supra); Delargy et al, New Phytol., 81:117-127 (1978); Harrison-Murray, Proc. Int. Plant Prop. Soc., 31:386-392 (1982)). The significance of lighting during the stock plant growth period and rooting of cuttings, with respect to the success of propagation, has been firmly established.
The technique of etiolation was used extensively in the 1920's (Reid, (1922 supra); Smith, (1924 supra); Knight et al, (1937 supra)). The additional pretreatment of banding stems to promote or maintain etiolation was added by Gardner in 1936. The procedure has remained essentially unchanged since that time. Etiolation is usually effected by covering the stock plant with black cloth or polyethylene, fastened over a structure enclosing the plant. Banding is accomplished with black tape, paper, tubing, aluminum foil (Biran et al, Sci. Hortic., 1:125-131 (1973): Davis et al, J. Environ. Hort., 1:96-98 (1983); Hare, USFS Res. Note No. SO-202. Southern For. Expt. Sta., New Orleans, La (1976)), or even black paste (Reuveni et al, Int. Soc. Hort. Sci. XXI Int. Hort. Congress., 1:1348 (1982)).
Interest in the practical uses of etiolation and the physiologicalanatomical basis for the etiolation effect has increased steadily since Frolich, (1961 supra), working with avocado, used etiolation to propagate experimental materials.
Perhaps the greatest contribution toward the development of a practical system for etiolation and banding has come from the efforts of researchers at the East Malling Research Station, Kent, England. From their work we have gained insight into the applicability of these pretreatments to a range of plant materials. Refinements in the process have been developed at East Malling, such as the use of heavy shade instead of complete darkness, ventilation of the shading structure, greening shoots for specified periods of time before taking cuttings, and comparisons of etiolation with banding as an additional pretreatment.
Methods of vegetative plant propagation such as mound layering (stooling) and air layering are based on the exclusion of light from the area to be rooted for a time before taking cuttings (Ryan (1969 supra)). The standard practice of placing cuttings in an opaque medium during rooting also benefits from the exclusion of light (Frolich (1961 supra)). The technique of stooling after etiolation has been used extensively by Frolich in his work with avocado. The procedure involves allowing shoots to extend in a dark chamber to a length of 3 inches before they are placed in a tarpaper collar filled with vermiculite to exclude light (Frolich, Calif. Avocado Soc. Yearbook, pp 136-138 (1951); Frolich, (1961 supra); Frolich et al, Calif. Avocado Soc. Yearbook, 55:97-109 (1972)). Schmidt, Int. Soc. Hort. Sci. XXI Int. Hort. Congress., 2:1785 (1982) compared the methods of etiolation, banding, and stooling in the propagation of Tilia tomentosa `Szeleste` and found the promotion of rooting to be due to the etiolation effect, and not the other effects of stooling (moisture, temperature).
Other pretreatments which have been used successfully with etiolation include hedging to induce shoot vigor (Delargy et al, (1978 supra); Mukherjee et al, J. Hort. Sci., 42:83-87 (1967)), ring barking (Delargy et al, (1978 supra)), and defoliation of the cutting (Howard, Plant Propagation. Rep. E. Malling Res. Stn. for 1983. (1984)).
Investigations on the practical level have offered much food for thought on the usefulness of etiolation and banding pretreatments.
The first study on the importance of synchronizing etiolation with the development of shoots came from Reid, (1922 supra), who compared etiolation periods of 14 and 28 days. The latter yielded shoots which were noticeably more etiolated, though the shorter treatment alone promoted rooting to 80%. Smith, (1924 supra) observed that shoots etiolated for too long a time were weakened, and tended to perish easily in the rooting environment. Gardner, (1936 supra) applied etiolation to apple trees just before bud break, and banded shoots for up to one year before taking cuttings, still noting a tremendous increase in rooting. The rooting response of etiolated shoots followed a pattern which Gardner observed in woody cuttings in general: good through August, and then poor until bud break the next spring (Gardner, (1936 supra)). Etiolation need only be applied for a length of time sufficient to yield usable shoots for banding or taking cuttings (Delargy et al, (1978 supra): Howard, Plant propagation. Rep. E. Malling Res. Stn. for 1978 (1979)). Etiolation has even been applied up to 2 weeks after bud break, with no decrease in effectiveness (Howard, Plant propagation. Rep. E. Malling Res. Stn. for 1981 (1982)).
The absence of chlorophyll, which is characteristic of etiolation, has no bearing on rooting success (Ryan, (1969 supra); Howard, Plant propagation. Rep. E. Malling Res. Stn. for 1979 (1980)). In general greening occurs rapidly while the rooting response declines very slowly upon exposure to light (Harrison-Murray et al, Int. Soc. Hort. Sci. XXI.sup.st Int. Hort. Congress, Hamburg. Abst. 1281 (1982)). It has been shown that in some cases 3 to 6 weeks greening after etiolation had no effect on rooting (Howard, (1980 supra); Schmidt, (1982 supra)), while up to 9 months in the light only decreased rooting to a level 40% above that of non-etiolated shoots (Howard, (1982 supra)). Important evidence regarding the effect of greening on rooting came from a study of an apple explants, which showed that after 2 weeks of darkness greening for 0 to 4 days had no effect on rooting, while 8 to 16 days in the light markedly decreased rooting. The response of rooting to light seems to be exaggerated in the in vitro situation.
Questions about the etiolation effect on rooting often turn to changes other than light in the environment surrounding the shoots. Howard, Plant propagation. Rep. E. Malling Res. Stn. for 1982 (1983) demonstrated that ventilating or using reflective covers to reduce the temperature under an etiolation structure could decrease the enhancement of rooting by etiolation. Work completed the following year (Howard, (1984 supra)), however, indicated that root number, not rooting percentage, was actually affected. Temperatures under black plastic ran on the average only 5 C above uncovered controls, while temperatures under clear plastic rose 13 C (Howard, (1984 supra)). Humidity, with minimal ventilation, did not increase under plastic coverings. An interesting observation was that even clear polyethylene reduced light transmission by 30%, and increased the rooting of M.9 apple rootstock by 10% over that of the controls.
A very important contribution toward making etiolation a more practical technique was the evidence that the complete exclusion of light during the growth of the shoot is not requisite to the stimulation of rooting. The use of 50% Saran shading to etiolate stock plants of Dahlia improved rooting from 7 to 75 percent (Biran et al, (1973 supra)), while 96% shade promoted the rooting of Tilia as well as 100% shade (Schmidt, (1982 supra)). The East Malling group tested a range of shading and found no decrease in rooting with up to a 20% transmission of light; even 70% shade (30% light transmission) still promoted rooting to 25% over that of the light grown controls (Howard, (1982, 1983, 1984 supra)).
The primary benefit of using partial versus complete shade is that shoots grown with some light incident upon them are hardier and thus better able to resist infection by Botrytis (Howard, (1983, 1984 supra)) and the scorching which a premature exposure to full sunlight can produce in etiolated shoots, which are lacking in protective pigmentation (Gardner, (1936 supra): Howard, (1984 supra)). In terms of production scheduling cuttings may be taken up to two weeks earlier if the recovery from etiolation is not needed (Howard, (1983 supra)). The alternative to shading has been to accustom etiolated shoots to light by uncovering them stepwise over a week or so before removing the shade enclosure entirely (Howard, (1980 supra)).
The observation that any reduction of light intensity can increase rooting brings into consideration a number of reports dealing with the growth of stock plants under light of varying intensity. In testing of stock plant irradiances in the range of 7 to 68 W*m-.sup.2 growing stock plants under lower irradiance was found to improve the rooting of Pisum (Anderson et al, Acta Hortic., 54:33-38 (1975): Hansen et al, Physiol. Plant., 32:170-173 (1974)), Hedera helix (Poulsen et al, Physiol. Plant., 49:359-396 (1980)), Malus (Christensen et al, Sci. Hort., 12:11-17 (1980)), Forsythia (Loach et al, Sci. Hort., 10:217-230 (1979)), and Vaccinium (Waxman, Plant Prop. Soc., 15:154-158 (1965)). In only a few instances has higher irradiance resulted in increased rooting, and then only root number was increased, rooting percentages being 100% in each case (Fisher et al, Sci. Hort., 7:171-178 (1977); Borowski et al, Sci. Hort., 15:245-253 (1981) - Chrysanthemum, Eliasson, Physiol. Plant., 43:13-18 (1978) - Pisum). The in vitro studies done with irradiance and rooting, in which other influences such as photoperiod, humidity, light quality, temperature and growing media are held constant, represent important insights into the effects lighting has on stock plants and the subsequent inhibition of root initiation (Anderson et al, (1975 supra); Hansen et al, (1974 supra): Christensen et al (1980 supra)).
The importance of using opaque banding material to maintain an etiolated section of stem, or in the case of blanching to exclude light from a section of stem grown in the light, has been well established by work done since Gardner's initial report in 1936 (Biran et al, (1973 supra): Delargy et al, (1978 supra): Delargy et al, New Phytol., 82:341-347 (1979): Harrison-Murray, (1982 supra); Schmidt, (1982 supra); Howard, Plant propagation. Rep. E. Malling Res. Stn. for 1976 (1977); Howard, Plant propagation. Rep. E. Malling Res. Stn. for 1977 (1978): Howard, Plant propagation. Rep. E. Malling Res. Stn. for 1980 (1981): Howard, (1979 to 1984 supra)).
Gardner, (1936 supra), in his work with apple, experimented with banding developing shoots close to the growing tip, or 1, 2, and 3 inches behind the tip. He left these bands on as the shoot matured, taking cuttings the next spring. Present methods commonly involve taking summer softwood cuttings, considerably shortening the time required for production. Banding very close to the developing tip gave the greatest response, while rooting response fell off as banding was applied further away from the shoot tip (Gardner, (1936 supra)). This indicates a need to exclude light early in the ontogeny of the shoot, before the cells derived from the shoot meristem have differentiated substantially. Gardner envisioned etiolation as an effective alternative to banding so closely to the easily damaged shoot tip, in which the exclusion of light could, from the time of bud break, influence the growth and differentiation of the shoot. Banding could then be applied further away from the shoot tip without endangering the growth of the apical meristem.
Howard, (1982 supra) also noted that blanching the stem immediately below the apex was nearly as effective as etiolation, in contrast to basal blanching which in previous studies proved ineffective in apple (Howard, (1981 supra)). Frolich's method of maintaining the etiolation effect was similar in that the daily addition of vermiculite to the tube surrounding the growing shoots kept light from the stem immediately below the apex (Frolich, (1951, 1961 supra)). Herman, Diss. Purdue Univ. Ph.D. DAI 28:B P0494 (1967) found that blanching was less effective than etiolation in enhancing the rooting of Phaseolus and Hibiscus, which could relate to the position of the band on the stem.
Very few reports of work with banding have indicated the width of band used. Delargy et al, (1979 supra) compared 2.5, 5, and 7.5 cm bands, and showed that 5 and 7.5 cm bands performed equally well, being 15% better than the 2.5 cm band, and 70% better than the control. Gardner did not experiment with varying band widths but had success with 2.5 to 3 inch (6.3 to 7.6 cm) width insulation tape (Gardner, (1936 supra)).
The same questions concerning the precise nature of the etiolation effect on rooting can be asked of banding. Though several studies have shown that clear plastic bands fail entirely to promote rooting, while opaque bands work quite well (Krul, (1968 supra): Kawase et al, (1980 supra); Delargy et al, (1979 supra)), no comparisons of clear banding at all, or measurements of the environmental conditions existent under black versus clear bands have been reported. Davis et al, (1983 supra) used aluminum foil to blanch a number of rhododendron cultivars and only 2 of 11 cultivars showed any increases in rooting. These disappointing results might be attributed to a need for prior etiolation, or a decrease in effectiveness when a reflective banding material is used. Biran et al, (1973 supra) shaded stock plants of Dahlia variabilis before banding with aluminum foil and achieved marked improvements in rooting.
The most apparent changes associated with the etiolation of shoots are those relating to the anatomy of the rooting zone in the resulting cuttings. The pronounced effects of etiolation include a lack of chlorophyll (equated with a characteristic chlorotic appearance), increases in internodal length, increased succulence, and decreased mechanical strength of stem tissues. A number of researchers have attributed a role for these changes in the stimulation of rooting by etiolation (for reviews see Stoutemeyer, Proc. Int. Plant Prop. Soc., 11:252-260 (1961), and Hess, (1969 supra)).
As far back as the 1920's gross anatomical changes associated with etiolation were being correlated with improved rooting. Reid, (1922 supra) found that etiolated shoots of camphor showed no traces of lignification, a possible mechanical barrier to adventitious rooting (Beakbane, Nature, 192:954-955 (1961); Beakbane, Proc. Int. Plant Prop. Soc., 19:192-201 (1969)), before the sixth stem node, as opposed to light grown stems which were lignified up to the third node. Etiolated stems also had less developed cell walls, and lacked the continuous fiber sheath found in light grown tissues (also see Smith, (1924 supra)--clematis, Blackie et al, (1926 supra)--camphor, Bid et al, Acta Hort., 24:77-81 (1972) - mango). Doud et al, J. Amer. Soc. Hort. Sci., 102:487-491 (1977) found much less sclerification in etiolated stems of Malus clones, which also rooted better than light grown stems. They established a strong negative correlation between degree of sclerification and rooting response. To the contrary, both Christensen et al, (1980 supra) and Raviv, Ph.D. thesis, Hebrew Univ. of Jerusalem (1981) concluded that, in their systems the presence of a fiber sheath had no direct effect on rooting. Hartmann et al, Plant Propagation: principles and practices. 4.sup.th edition (1983) agree with these researchers; even though plants with lower amounts of lignification and fiber often root more easily, these anatomical changes cannot be considered the primary determinants in rooting. In carnation stem cuttings, for example, roots which initiate within a thick fiber sheath will merely grow down before emerging. Beakbane, (1961 supra) noted that such mechanical barriers to rooting were not the only possibilities for decreased rooting because in poor rooting cuttings initials often would not form even to the inside of fiber sheaths.
One of the most cited arguments for anatomical involvement came from Frolich, (1961 supra) who showed that in avocado (Persea americana) there was no transmission of a rooting influence either up or down the stem from an etiolated section bordered by light grown tissue. This was supported by observations of Doss et al, Acta Hort., 112:77-84 (1980) on the in vitro rooting of shoots of Rubus idaeus `Meeker` which had been etiolated or blanched. While the length of the etiolated section was varied roots consistently initiated only in the etiolated sections. Delargy et al, (1978 supra) applied bands at positions on the stem adjacent to and distal from the cutting base, and found that rooting occurred twice as well when the rooting zone was also the etiolated zone, as opposed to being adjacent to the etiolated zone.
Contrary evidence for a transmissable etiolation effect has been presented by Kawase et al, (1980 supra), working with hypocotyls of Phaseolus vulgaris wrapped in black or clear plastic. In this rather elegant system roots formed only under black, not under clear, plastic. In an experiment using multiple bands placed along the stem, they found that the presence of a band would stimulate the rooting in a band immediately below, though not under a band basally removed by 4 cm. This suggests that there is a transmission of root promoting influence. The short distance over which the influence acted may indicate that the substances diffused from the banded area above to that below, as opposed to being actively transported. Howard, (1983 supra) reported that etiolation immediately distal to a basal opaque band increased root number under the band from 4.9 to 21.7 roots per cutting, indicating that substances produced in etiolated tissues can move basipetally and influence rooting in other areas shaded from light. More research will be needed to solve this particular controversy.
Other anatomical factors which have been studied include the effects of etiolation on internodal length. The longer nodes characteristic of etiolated stems have been correlated with higher rooting in pea (Veierskov, Physiol. Plant., 42:146-150 (1978)). Two factors which may deserve more consideration in the future include (1) the effect of etiolation on the proportion of undifferentiated parenchyma in the pericycle and phloem ray systems of the stem, both sites of root initiation (Snyder, Proc. Int. Plant Prop. Soc., 12:43-47 (1962)), and (2) light effects on the suberization of stem tissues, which has recently been correlated with the rooting success of 19 Australian woody plant species, acting perhaps as an anatomical barrier to root emergence (Williams et al, Aust. J. Bot., 32:363-366 (1984}). Etiolation and banding promote increases in the proportion of stem tissues occupied by undifferentiated parenchyma (Stoltz et al, Proc. Amer. Soc. Hort. Sci., 84:734-743 (1966); Stoltz et al, Proc. Amer. Soc. Hort. Sci., 84:744-751 (1966): Herman, (1967 supra); Schmidt, (1982 supra)), which is known to be an intermediate in the initiation of adventitious roots (Hartmann et al, (1983 supra)), and could be associated with the increase in herbaceous character caused by etiolation and correlated with improved rooting (Frolich, (1961 supra); Bid et al, (1972 supra); Biran et al, (1973 supra); Christensen et al, (1980 supra)).